载体pLVX-Tight-Puro

pLVX-Tight-Puro is a tetracycline (Tet)-inducible, lentiviral expression vector designed to express a gene of interest under the control of PTight, a modified Tet-responsive promoter. PTight consists of a modified minimal CMV promoter, and seven direct repeats of a 36 bp regulatory sequence that contains the 19 bp tet operator sequence (tetO; 1). This vector is designed to be used with our Lenti-XTM Tet-On® Advanced and Tet-Off® Advanced Inducible Expression Systems (Cat. Nos. 632162 and 632163). These systems provide the inducible gene expression strategy of Gossen & Bujard, with major improvements described by Urlinger, et al. (2-6), in a lentiviral format.

pLVX-Tight-Puro contains all of the viral processing elements necessary for the production of replication-incompetent lentivirus, as well as elements to improve viral titer, transgene expression, and overall vector function. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) promotes RNA processing events and enhances nuclear export of viral and transgene RNA (7), leading to increased viral titers from packaging cells, and enhanced expression of your gene of interest in target cells. In addition, the vector includes a Rev-response element (RRE), which further increases viral titers by enhancing the transport of unspliced viral RNA out of the nucleus (8). Finally, pLVX-Tight-Puro also contains a central polypurine tract (cPPT) element that increases nuclear importation of the viral genome during target cell infection, resulting in improved vector integration and more efficient transduction (9)..

In addition to lentiviral elements, pLVX-Tight-Puro contains a puromycin resistance gene (Puror) under the control of the murine phosphoglycerate kinase (PGK) promoter (PPGK) for the selection of stable transductants. The vector also contains a pUC origin of replication and an E. coli ampicillin resistance gene (Ampr) for propagation and selection in bacteria.

pLVX-Tight-Puro is a lentiviral expression vector that allows tightly regulated, doxycycline-controlled expression of a gene of interest. In order to function, the system requires the presence of a tetracycline-controlled transcriptional activator (rtTA Advanced or tTA Advanced) supplied by either pLVX-Tet-On Advanced or pLVX-Tet-off Advanced lentiviruses. Before pLVX-Tight-Puro can be transduced into cells, it must be transfected into 293T packaging cells with our Lenti-X HTX Packaging System (Cat. Nos. 631247 and 631249). This packaging system allows you to safely produce infectious, replication-incompetent, VSV-G pseudotyped lentiviral particles that can infect a wide range of cell types, including non-dividing and primary cells (10).